This is a revised application to examine the mechanism of RNA encapsidation during replication of Sin nombre virus (SNV). SNV is a Category A pathogen and a member of the hantavirus genus in the Bunyaviridae family. We anticipate that the general principles of RNA encapsidation that take place during SNV infection will be applicable to the other members of the hantavirus genus and likely to the entire Bunyaviridae family. Since submission of the original proposal we have obtained substantial additional preliminary data. These preliminary data lead us to two major hypotheses. First, the terminal viral panhandle RNA structure is the cis-acting encapsidation sequence recognized by the viral nucleocapsid protein. Second, the nucleocapsid protein is a nonspecific RNA chaperone required for the generation of biologically functional higher order viral RNA structure. The work proposed in this R21 application will characterize the interaction between the nucleocapsid and the viral RNA panhandle, and also examine the mechanism of action of the RNA chaperone activity of the nucleocapsid protein. We anticipate that the general properties of the viral RNA and nucleocapsid protein of SNV be applicable to other members of the hantavirus genus and also to other members of the Bunyaviridae family. Previous work from our lab focused on lentivirus encapsidation, RNA structure, and the cis- and transcomponents required for encapsidation of dimeric HIV RNA. Our past experience with this positive stranded RNA virus will likely be useful as we investigate encapsidation of negative stranded hantavirus RNA. We have generated some preliminary data concerning facets relevant to hantavirus RNA encapsidation, and some of these data are described in this proposal. However, since we have only recently embarked on the hantavirus system and do not yet have funding for our proposed work, it seemed appropriate to apply for an R21. Following our preliminary investigations, our longer range plans include trying to generate a comprehensive picture of RNA encapsidation for SNV, and other members of the Bunyaviridae, and to seek funding through the RO1 mechanism. In addition to characterizing the features of the biology of hantavirus encapsidation, our longer term goals will also include attempting to make use of this information by devising screening strategies for these viruses and perhaps by generating hantavirus-based vectors for various applications.